This chapter examines technical considerations associated with the use of 15N in nucleic acid stable isotope probing (SIP) experiments, discusses concerns that should be considered prior to undertaking a 15N-labeling experiment, and provides an overview of different applications of 15N-SIP. The preferable approach is to use quantitative PCR (qPCR) to determine the number of 16S rRNA genes in each gradient fraction. The gene target for qPCR analysis can vary by application, and it may be desirable to use universal 16S rRNA gene-targeted primers or primers that are specific to individual domains, individual subgroups, or genera. In DNA purified by secondary gradient fractionation, nif H genes similar to Methylosinus represented 53% of those recovered while Methylocystis-like sequences represented 17% of those recovered. However, in an experiment in which 15N2-DNA-SIP was used to examine nitrogen-fixing methanotrophs in soil. There are several reasons why 15N-DNA-SIP represents an appealing method for examining nitrogen (N2)-fixing organisms. First, incubations can be carried out at realistic concentrations of substrate, as air can be evacuated from sealed containers and replaced with simulated air containing 15N2. Second, since nitrogen fixation is inhibited in the presence of mineral forms of nitrogen, problems associated with isotope dilution can largely be ignored. Experiments will need to be performed with pure cultures and environmental samples to determine whether 15N-RNA-SIP can be used effectively in microbial ecology studies.

Effect of varying the atom% 15N of NH4Cl on the CsCl buoyant density of DNA from E. coli grown in minimal media. Reprinted from Buckley et al. (2007a, Supplementary Materials) with permission of the publisher.

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FIGURE 1.

Effect of varying the atom% 15N of NH4Cl on the CsCl buoyant density of DNA from E. coli grown in minimal media. Reprinted from Buckley et al. (2007a, Supplementary Materials) with permission of the publisher.

Expected relationship between genome G+C content and buoyant density in a CsCl gradient for unlabeled DNA and DNA that is partially or completely labeled with 15N (see legend). The shaded region of the chart represents the range of densities over which unlabeled DNA would be expected to occur based biologically meaningful values of genome G+C content (30% to 80% G+C content). Reprinted from Buckley et al., (2007a) with permission of the publisher.

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FIGURE 2.

Expected relationship between genome G+C content and buoyant density in a CsCl gradient for unlabeled DNA and DNA that is partially or completely labeled with 15N (see legend). The shaded region of the chart represents the range of densities over which unlabeled DNA would be expected to occur based biologically meaningful values of genome G+C content (30% to 80% G+C content). Reprinted from Buckley et al., (2007a) with permission of the publisher.

Theoretical relationship between the length of a DNA fragment with a buoyant density of 1.71 g ml-1 and the time required for that DNA fragment to reach equilibrium in a CsCl density gradient of mean density of 1.69 g ml-1 formed in a TlA110 rotor at 55 k rpm (164,000 × g). Reprinted from Buckley et al. (2007a, Supplementary Materials) with permission of the publisher.

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FIGURE 4.

Theoretical relationship between the length of a DNA fragment with a buoyant density of 1.71 g ml-1 and the time required for that DNA fragment to reach equilibrium in a CsCl density gradient of mean density of 1.69 g ml-1 formed in a TlA110 rotor at 55 k rpm (164,000 × g). Reprinted from Buckley et al. (2007a, Supplementary Materials) with permission of the publisher.

T-RFLP analysis of 16S rRNA genes can be performed to examine the buoyant density distribution of individual TRFs in primary gradient fractions. Symbols correspond to a TRF of 144 bp length generated by 16S rRNA gene TRFLP analysis of DNA from soil incubated either in artificial air (○) or in artificial air containing 15N2 (●). TRF peak height was normalized as a function of the maximum peak height in each gradient. Reprinted from Buckley et al. (2007b) with permission of the publisher.

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FIGURE 5.

T-RFLP analysis of 16S rRNA genes can be performed to examine the buoyant density distribution of individual TRFs in primary gradient fractions. Symbols correspond to a TRF of 144 bp length generated by 16S rRNA gene TRFLP analysis of DNA from soil incubated either in artificial air (○) or in artificial air containing 15N2 (●). TRF peak height was normalized as a function of the maximum peak height in each gradient. Reprinted from Buckley et al. (2007b) with permission of the publisher.